1. Field of the Invention
The invention relates to a method and apparatus for performing contact x-ray microscopy using an optical, phase contrast microscope for inspecting and aligning a target prior to exposing the target to a source of soft x-rays.
2. Description of Related Art
Numerous efforts have been made in the past to examine small objects using x-rays. Early efforts have always encountered problems when making observations with x-rays especially for small objects which were especially difficult to cope with when living media was the target.
Perhaps the most relevant prior reference is U.S. Pat. No. 2,877,353 entitled X-RAY MICROSCOPE. It describes what is known as an x-ray shadow wherein a target is bombarded by x-rays and its shadow is recorded on an x-ray sensitive medium. The x-ray sensitive medium could be a television screen or a film or the like. U.S. Pat. No. 2,939,954 entitled X-RAY SHADOW MICROSCOPE also discloses another early effort to capture x-ray images of targets.
The following U.S. Pat. Nos. describe efforts to use television in the context of x-ray microscopes: 3,818,233; 3,743,845; 3,846,632 and 3,860,819. U.S. Pat. No. 3,818,233 also suggests the possibility of using an optical device in combination with an x-ray television microscope.
The following U.S. Pat. Nos. describe prior x-ray microscopes of more general relevance: 2,617,942; 2,754,425; 3,079,501; 3,143,651; and 4,317,036.
It is also possible to have the reverse of a microscope, namely a telescope. See, for example, U.S. Pat. No. 4,562,583 entitled SPECTRAL SLICING X-RAY TELESCOPE WITH VARIABLE MAGNIFICATION.
Sources of x-rays which might be employed for microscopes or for other purposes are described in U.S. Pat. Nos. 4,538,291 and 4,596,030 as well as in other prior art sources.
Descriptions of other devices for producing soft x-ray beams may be found, for example, in U.S. Pat. Nos. 4,704,718 entitled APPARATUS AND METHOD FOR GENERATING SOFT X-RAY LASING ACTION IN A CONFINED PLASMA COLUMN THROUGH THE USE OF A PICOSECOND LASER and U.S. Pat. No. 4,771,430 entitled ENHANCEMENT OF SOFT X-RAY LASING ACTION WITH THIN BLADE RADIATORS. While the use of soft x-ray lasers in the context of x-ray microscopy has been speculated about over the years, there is no known discussion of the use of such device in the specific apparatus and combination taught in this disclosure.
The following U.S. Pat. Nos. are of possible relevance in that they relate to x-ray imaging: 2,557,662; 2,559,972; 2,759,106; 3,407,296 and 4,253,154.
In addition to the foregoing patents, the following literature references may also be relevant:
1. M. Rousseau, Spectrophotometrie de Fluorescence en Microscopie, Bull. Microscopie Appl. 7, 92-94 (1957).
2. B. Chance, and B. Thorell, Localization and Kinetics of Reduced Pyridine Nucleotides in Living Cells by Microspectrofluorometry, Jour. Biol. Chem. 234, 3044-3050 (1959).
3. R. A. Olson, Rapid Scanning Microspectrofluorometer, Rev. Sci. Instr. 31, 844-849 (1960).
4. T. Caspersson, G. Lomakka, and R. Rigler, Jr., Regiestrierender Sedundarfluoreszenz Verschiedene Zellsubstanzen, Act. Histochem. Suppl. 6, 135-153 (1965).
5. B. Thorell, E. Kohen, and C. Kohen, Metabolic Rates and Intercellular Transfer of Molecules in Cultures of Human Glia and Glioma Cells, Med. Biol. (Helsinki) 56, 386-392 (1978).
6. G. H. I. Sloane, and C. N. Loesser, Spectroscopic Analysis of Carcinogenic Hydrocarbons in Biologic Interactions in vivo and in Vitro, Cancer Res. 23, 1555-1556 (1963).
7. P. Daudel, M. Croisy-Delcey, C. Alonso-Verduras, M. Duquesne, P. Jacquignon, P. Markovits, et al. P. Vigny, Etude par Fluorescence d'Acides Nucleiques Extraits de Cellules en Culture Traitees par le Methyl Benzo(a)anthracene, Comptes Rend. Acad. Sci. Paris 278, 2249-2252 (1974).
8. J. M. Salmon, E. Kohen, C. Viallet and F. Zajdela, A Preliminary Microscptrofluorometric Study of NAD(P)H Reduction i Dibenzo (a,e) Fluoranthrene-Treated Single Living Cells, Histochem 47, 291-302 (1976).
9. B. Thorell, Flow-Cytometric Monitoring of Intracellular Flavins Simultaneously with NAD(P)H Levels, Cytometry 4, 61-65 (1983).
10. A High Resolution Grating Microspectrofluorometer with Topographic Option for Studies in Living Cells, J. G. Hirschberg, et al., A.C.S. SYMPOSIUM SERIES No. 102 Multichannel Image Detectors, Yair Talmi, Editor. American Chemical Society, 1979.
11. S. Suckewer et al., Phys. Rev. Lett. 55, 1753 (1985).
12. S. Suckewer et al., Phys. Rev. Lett. 57, 1004 (1986).
13. S. Suckewer et al., physique, Supplement 10, Vol. 47 C6-23 (1986).
In the study of the functions of the living cell, one of the most successful approaches has been the use of monolayer cultures of cells which exhibit the fluorescence of naturally occurring coenzymes produced by the life processes of the cell especially in NADH and flavo proteins. Descriptions of the foregoing can be found in literature references numbered 1 through 5 above. Another highly successful application of fluorescence is in the detection and determination of the fate of foreign materials in a cell, known as xenobiotics, such as carcinogens and their poisons. Further discussions of the foregoing are findable in literature references number 6 through 9 referred to above. Both applications are made possible by the remarkable sensitivity of the fluorescence method where as little as one part in a quadrillionth of a mole can be detected.
In the prior art, the resolution available was generally limited to about 250 nm when visible light wavelengths were employed. In many experiments, however, especially where the mechanism of a cell's defense against xenobiotics is investigated, fluorescence was found to spread throughout the cell a few seconds after injection. The exact mechanism of fluorescent spreading remains obscure due to the limitation of the available resolution. Attempts have been made to obtain increased information by circling the injected cell or cells with a diamond, and later using an electron microscope. Unfortunately, this technique suffers from the problem that the cells must be desiccated and then shadowed with evaporated metal before placing them in the electron microscope. All time-resolution is lost by this process, and the cellular structure is, of course, altered thereby.
References number 11-13 above relate to sources of soft x-ray laser beams which might be advantageously employed with the present invention. The foregoing references further discuss the soft x-ray laser devices found in U.S. Pat. Nos. 4,704,718 and 4,771,430 previously described.
A useful overview of the state of the art of x-ray microscopes can be found in an article entitled "Soft-x-ray Microscopes", Physics Today, August 1985, pages 22-32.
The use of photoresists as applied to x-ray microscopes is discussed in an article entitled "Specimen Replication for Electron Microscopy Using X-Rays and X-Ray Resist", Journal of Applied Physics, Vol. 47, No. 3, March 1976, pages 1192-1193.
The measurement of calcium using soft x-rays is discussed in an article entitled "Absorption Microanalysis with a Scanning Soft X-Ray Microscope: Mapping the Distribution of Calcium in Bone" by J. M. Kennedy et al., Journal of Microscopy, Vol. 138, June 1985 pages 321-328.
Lastly, two additional useful background articles relating to the state of the art of high resolution microscopes include "Scanning X-Ray Microscope with 75-nm Resolution", by H. Rarback, et al., Rev. Sci. Instrum. 59(1), January 1988 pages 52-59 and "Some Experiences with X-Ray and Proton Microscopes", by Paul Horowitz, New York Academy of Sciences, 1978, pages 203-222.
Insofar as understood none of the forementioned references describes an apparatus or method for x-ray microscopy with an effective way to align a target, especially a target comprising living cells with an optical microscope prior to exposure by a soft x-ray beam.